This project will develop and characterize the application of RASL-Seq, a multiplexed targeted expression profiling technology that uses q sequencing read-out, to analysis of banked tissues. The technology detects hundreds of RNA targets at once in cell lysates, using oligo ligation, amplification, and next-generation sequencing for quantitation. This highly multiplexed assay will allow a simple, fixed tissue assay protocol that can be applied to different rodent tissues, while delivering a simple expression profiling report. The technology will promote toxicology research by enabling direct comparisons between expression profiles derived from compound screening in vitro to those observed in animals dosed with the same compounds. It will do so at a low cost/sample by multiplexing 20 to 100 or more samples/sequencing run, thus reducing the sequencing cost/sample to $6 from $600. We will optimize the lysis buffer/protocol for formalin-fixed paraffin-embedded (FFPE) tissues, design and test probes for degraded RNAs, build an assay panel for ~100 rodent genes of interest for animal testing in toxicology studies, and assess the performance of the assay on archived tissues from animals dosed with compounds with known impacts on gene expression. Performance measures will include reproducibility, dynamic range, fold difference detection, and correlation with organ-specific and compound-specific expression changes. If successful, the project will deliver a technology that will allow integration of in vitro and in vivo profiling, whch may provide enhanced compound response prediction, increasing the efficiency of animal testing, and also enable the development of reliable in vitro models which will lead to the use of fewer animals. Finally, the sensitivity of the RASL-Seq assay and gene expression read-out may permit sub-toxic adverse effects of compounds to be detected, permitting potential toxicity to be identified that is not observable by micro-histology.